Identification and Structural Elucidation of Galloylated B‐Type Procyanidins From <i>Rumex obtusifolius</i> L. Using Enzymatic Digestion, Phloroglucinolysis, and High‐Resolution Mass Spectrometry

Procyanidins from plant extracts demonstrate a broad spectrum of bioactive potential. In order to establish structure-activity relationships, it is necessary to fully determine the stereochemistry and substitution pattern. Phloroglucinolysis and nuclear magnetic resonance (NMR) are conventionally employed for the analysis of galloylated B-type procyanidins (PBgals). However, these methods require substantial quantities of the compound at a high degree of purity. In this work, five dimeric galloylated procyanidins isolated from Rumex obtusifolius L. roots were analyzed using a two-pronged strategy. Initially, identification and structure elucidation were performed through the implementation of a novel miniaturized enzymatic digestion with polygalacturonase to effectively remove gallic acid residues. Subsequently, the remaining degalloylated dimeric procyanidins were compared to procyanidin B1, B2, B3, B5, and B7 reference substances based on their retention times. Thus, the R/S-configurations at the C2 and C3 positions of both monomer units and the interflavan linkage were determined as either C4→C8 or C4→C6 in a single analysis. In addition, the position of galloylation was ascertained on the upper or lower unit via high-resolution MS with collision-induced dissociation (CID) and electron-activated dissociation (EAD) fragmentation. This innovative fragmentation technology has been demonstrated to preserve the labile bonds, such as the ester bond to gallic acid. Hence, it yields novel diagnostic fragments for structure elucidation. In silico fragmentation analysis revealed that the radical fragment m/z 440.0738 only fits galloylation on the upper unit, thereby validating the results of the CID. Furthermore, phloroglucinolysis was equally miniaturized and utilized as an independent method to corroborate the results of enzymatic digestion and LC-MS/MS. Using these methods, the structural configurations of the five isolated substances were successfully and consistently identified as procyanidin B2-3-O-gallate, procyanidin B1-3-O-gallate, B2-3'-O-gallate, procyanidin B7-3-O-gallate, and procyanidin B5-3'-O-gallate. To our knowledge, this is the first time that these PBgals are reported in R. obtusifolius.